Egg white processing

ABSTRACT

This disclosure relates to inexpensive and efficient methods of preparing egg white (e.g., obtained from eggs laid by transgenic chickens) for bulk chromatographic isolation of proteins (e.g., recombinant proteins) from the egg white, as well as method of filtering acidified egg white and methods of isolating proteins from the egg white.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application under 35 U.S.C. §120 ofU.S. patent application Ser. No. 15/903,714, filed Feb. 23, 2018, whichis a continuation of U.S. patent application Ser. No. 14/691,817, filedApr. 21, 2015, which claims priority to U.S. Provisional Application No.61/983,003, filed on Apr. 23, 2014, the contents of each of which arehereby incorporated by reference in their entirety.

BACKGROUND

Transgenic avians (e.g., transgenic chickens, quails or turkeys) are adesirable expression system for obtaining exogenous recombinant proteinsfor use in pharmaceutical or other commercial applications that requirelarge amounts of protein supply. A hen can lay up to 330 eggs per year,each containing 6.5 grams of protein. About 3.5 grams of the totalprotein is from egg white, of which 90% is accounted for by sevendifferent proteins; the ovalbumin alone accounts for 2 grams of eggwhite protein (about 50% of the egg white protein). Currently, theaverage exogenous gene product derived from oviduct specific expressionof a transgene and recovered from the egg white is known to be about5-10 mg per egg. Advantages of exogenous protein production in chickeneggs include short generation times and prolific rates of reproductionvia artificial insemination. Various proteins have been expressed ineggs of transgenic chickens. See, e.g., U.S. Pat. No. 6,730,822 and U.S.Publication No. 2006/0015960.

Many exogenous therapeutic proteins (e.g., recombinant human proteinssuch as cytokines (e.g., erythropoietin, granulocyte colony-stimulatingfactor (GC-SF), interferons, and granulocyte-macrophagecolony-stimulating factor (GM-CSF)), antibodies, and various humanlysosomal enzymes) are of interest to the pharmaceutical industry. Thetherapeutic proteins can readily be obtained in significant quantitiesfrom, for example, egg white of transgenic chickens. Traditional methodsof isolating exogenous proteins from the egg white, however, often relyon use of immunoaffinity procedures or other procedures only suitablefor small scale production (e.g., involving total egg white volume of 5L at most). For a large-scale protein production, such a procedure isnot practical based on costs, labor, and time.

SUMMARY

This disclosure is based on the unexpected discovery that adding a smallamount of an acidic buffer (e.g., in a single bolus injection) to a poolof egg white (e.g., obtained from eggs laid by transgenic avian such aschickens, quails and turkeys) of industrial scale (e.g., having a volumeof at least 10 liters) can prepare the egg white for bulkchromatographic isolation of recombinant proteins such as humantherapeutic proteins from the egg white without the need of diluting theegg white. Such a method can significantly reduce the amount of eggwhite materials subject to the downstream isolation/purificationprocesses (e.g., the amount of the columns used in chromatographicisolation), thereby significantly reducing the costs, labor, and time ofisolating recombinant proteins from egg white. Accordingly, the methodsdescribed in the present disclosure can greatly improve the efficiencyof egg white preparation for a large, industrial scale of therapeuticprotein production.

In one aspect, this disclosure features a method of preparing egg whitefor bulk chromatographic processing that includes the steps of: (1)adding an acidic buffer comprising an acidic agent to a pool of eggwhite, the acidic buffer being from about 0.5 wt % to about 5 wt % perkilogram of the egg white; and (2) mixing the acidic buffer and the eggwhite to form a mixed egg white having a pH of from about 5 to about6.5.

In another aspect, this disclosure features a method of isolating arecombinant protein from egg white that includes the steps of: (1)providing a pool of egg white containing a recombinant protein, the poolhaving a volume of at least about 10 liters; (2) adjusting the pH of theegg white to from about 5 to about 6.5, in which the conductivity of thepH-adjusted egg white is from about 8 mS/cm to about 20 mS/cm; (3)filtering the egg white to form a solution (i.e., a clear solution); and(4) isolating the recombinant protein in the egg white by columnchromatography.

In still another aspect, this disclosure features a method of filteringacidified egg white that includes the steps of: (1) passing apre-treatment buffer having a conductivity between about 8 mS/cm andabout 20 mS/cm through a filter; and (2) passing egg white having a pHfrom about 5 to about 6.5 through the filter to obtain a filtered eggwhite.

Embodiments can include one or more of the following features.

The acidic agent can be selected from the group consisting of aceticacid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid,hydrofluoric acid, hydrobromic acid, perchloric acid, citric acid, boricacid, tartaric acid, lactic acid, formic acid, oxalic acid, uric acid,and barbituric acid.

The acidic buffer can further include from about 5M to about 6M (e.g.,about 5.7 M) sodium acetate.

The acidic buffer can be from about 0.5 wt % to about 2 wt % (e.g., fromabout 0.7 wt % to about 1.5 wt %, from about 0.9 wt % to about 1.4 wt %,or from about 1.2 wt % to about 1.3 wt %) per kilogram of the egg white.

The acidic buffer can have a pH from about 4 to about 6.5 (e.g., about4, about 4.5, about, 5, about 5.5, about 6.0 or about 6.5).

After mixing the acidic buffer with the egg white, the pH of the mixedegg white can be from about 5 to about 6.5. In some embodiments, the pHof the mixed egg white is from 5 to about 6.3 (e.g., from about 5.7 toabout 6.3, from about 5.8 to about 6.2, from about 5.9 to about 6.1, orabout 6). In some embodiments, the pH of the mixed egg white is a valuesuch that the mixed egg white is rendered least viscous.

The egg white can be mixed for at least about 1 hour and/or at atemperature from about 2° C. to about 25° C.

The egg white pool can have a volume of at least about 10 liters (e.g.,at least about 50 liters).

The acidic buffer can be added to the egg white pool in a single bolusinjection and/or at a rate of at least about 1 L/minute.

The addition of the acidic buffer to the egg white and the mixing of theegg white can be performed concurrently.

The method can further include a step of allowing the mixed egg white tosettle such that the egg white separates into top, middle and bottomlayers. In such embodiments, the method can further include a step ofisolating the middle layer. In some embodiments, the method can furtherinclude filtering the middle layer after the isolation step. Thefiltering can include passing at least a portion (e.g., all) of themiddle layer through a filter having an average pore size ranging fromabout 0.1 μm to about 100 μm. In some embodiments, the filtering caninclude passing at least a portion of the middle layer through aplurality of filters.

The method can further include filtering the mixed egg white withoutallowing the mixed egg white to settle. In such embodiments, thefiltering step can include filtering the mixed egg white through afilter having an average pore size ranging from about 0.1 μm to about100 μm. In some embodiments, the filtering step can include one or moresubsequent filtering steps following an initial filtering of the mixedegg white, the one or more subsequent filtering steps using one or morefilters having an average pore size ranging from about 0.1 μm to about40 μm.

The method can further include a centrifugation step after the mixingstep, in which the mixed egg white is centrifuged to separateprecipitates containing ovomucin-lysozyme complexes from supernatant.

The egg white can include a recombinant therapeutic protein exogenous toegg white.

The acidic buffer can have a conductivity from about 8 mS/cm to about 40mS/cm.

The acidic buffer can be from about 0.5 wt % to about 5 wt % perkilogram of the egg white.

A pre-treatment buffer can be used to prepare filters for passingacidified egg white. The pretreatment buffer can have a pH substantiallysimilar to or the same as the pH of the egg white, ranging from about5.0 to about 6.5. For example, the pre-treatment buffer can have a pHfrom about 5.9 to about 6.1 (e.g., about 6).

The pre-treatment buffer can include sodium phosphate and sodiumchloride.

The pre-treatment buffer can have a conductivity from about 10 mS/cm toabout 20 mS/cm.

The acidified egg white can have a conductivity from about 8 mS/cm toabout 20 mS/cm.

The filter can have a filtration medium area of at least about 8 m².

The filter can have an average pore size from about 0.1 μm to about 100μm.

The egg white can be passed through the filter under a differentialpressure less than about 30 psi (e.g., less than about 15 psi).

Embodiments can have the following advantages.

In general, chromatographic columns used in isolation/purification ofexogenous protein from egg white are very costly, which can be one ofthe key limiting factors in commercial and industrial scale productionof recombinant proteins from egg white. Because only a small amount ofan acidic buffer is used to obtain acidified egg white, the amount ofthe acidified egg white used to obtain purified therapeutic proteins issignificantly reduced (i.e., at least 3 to 4-fold less than conventionaldilution methods). As a result, time consumed for loading the acidifiedegg white to the subsequent columns is also significantly reduced, whichpermits rapid protein production in an industrial process. In addition,because some therapeutic proteins are sensitive to the environmentexposed during the egg white preparation and isolation/purificationprocesses, minimizing the time spent in the preparation andisolation/purification processes by reducing sample volume is highlyadvantageous for commercial production that typically involves an eggwhite volume of 500L or more. In sum, processing time, materials andlabor can be greatly minimized by using the methods described herein.

Other features, objects, and advantages will be apparent from thedescription and drawings, and from the claims.

DETAILED DESCRIPTION

This disclosure relates to efficient methods of preparing egg white(e.g., obtained from eggs laid by transgenic chickens) for bulkchromatographic isolation of proteins (e.g., recombinant proteins) fromthe egg white, as well as methods of isolating proteins from the eggwhite. The egg white prepared by the methods described herein isgenerally in the form a homogeneous, low viscosity solution that issuitable for bulk chromatographic processing.

In some embodiments, egg white used as a starting material of themethods described herein can include a recombinant protein (e.g., arecombinant therapeutic protein) exogenous to the egg white. Exemplaryrecombinant proteins include cytokines such as GC-SF, GM-CSF,erythropoietin, and interferons such as interferon-α or interferon-β;human lysosomal enzymes; immunoglobulins (e.g., antibodies); andstructural proteins. Other exemplary recombinant proteins that can beisolated from bulk chromatographic processing have been described, e.g.,in U.S. Application Publication No. 2009/0299037.

In general, the methods of preparing egg white described herein include(1) adding an suitable amount of an acidic buffer (e.g., from about 0.5wt % to about 5 wt % per kilogram of the egg white) containing an acidicagent to a pool of egg white (e.g., having a volume of at least about 10liters); and (2) mixing the acidic buffer and the egg white to form amixed egg white having a suitable pH (e.g., a pH ranging from about 5 toabout 6.5).

In general, the egg white pool used in the methods described herein isat an industrial scale and has a relatively large volume. For example,the egg white pool can have a volume of at least about 10 liters (e.g.,at least about 50 liters, at least about 100 liters, at least about 200liters, at least about 300 liters, at least about 400 liters, at leastabout 500 liters, at least about 600 liters, at least about 700 liters,at least about 800 liters, at least about 900 liters, at least about1,000 liters, at least about 1,500 liters, at least about 2,000 liters,at least about 3,000 liters, at least about 4,000 liters, at least about5,000 liters, at least about 10,000 liters, or at least about 20,000liters). Methods of preparing egg white as a starting material to beused in the methods described herein have been described, e.g., in U.S.Application Publication No. 2009/0299037.

The acidic agent in the acidic buffer can generally be any suitable acid(e.g., an organic acid or an inorganic acid). Exemplary acidic agentsinclude acetic acid, hydrochloric acid, sulfuric acid, nitric acid,phosphoric acid, hydrofluoric acid, hydrobromic acid, perchloric acid,citric acid, boric acid, tartaric acid, lactic acid, formic acid, oxalicacid, uric acid, and barbituric acid. In some embodiments, a combinationof two or more (e.g., three or four) acids can be used as the acidicagent in the acidic buffer.

In some embodiments, the acidic buffer can include one or more salts(e.g., alkaline salts). An example of such a salt can be sodium acetate.In some embodiments, the salt used in the acidic buffer can be a salt ofthe acidic agent used in the acidic buffer. In other embodiments, thesalt used in the acidic buffer can be a salt of an acid different fromthe acidic agent used in the acidic buffer. In some embodiments, theacidic buffer can include from about 5M to about 6M (e.g., about 5.7 M)of a salt (e.g., sodium acetate). Without wishing to be bound by theory,it is believed that using a salt having such a concentration can resultin an acidic buffer having a suitable amount of buffering content. Ifthe buffering content is too high, the acidic buffer may becomeflammable and/or corrosive, rendering the buffer unsafe to maintain,handle, and/or store. If the buffering content is too low, a largervolume of the acidic buffer might be needed to adjust the pH of the eggwhite to the target value, thereby reducing the efficiency of the eggwhite preparation process, as well as the downstream proteinisolation/purification processes.

In general, the amounts of the acidic agent and the salt used in theacidic buffer can vary depending on the desired pH of the acidic buffer.In some embodiments, the acidic buffer can have a pH from about 4 toabout 6.5 (e.g., from about 4 to about 5 or from about 4 to about 4.5).For example, the acidic buffer can have a pH of about 4 or a pH of about4.5.

The acidic buffer can be formed by any suitable methods known in theart. For example, the acidic buffer can be formed by adding an acidicagent to a solution containing a base to adjust the pH of the solutionto a desired value. For example, glacial acetic acid solution can beadded to an appropriate amount of NaOH to obtain an acidic buffercontaining 5.7M sodium acetate.

In general, the amount of the acidic buffer relative to that of the eggwhite is small. For example, the acidic buffer can be from about 0.5 wt% to about 5 wt % (e.g., from about 0.5 wt % to about 2 wt %, from about0.7 wt % to 1.5 wt %, from about 0.9 wt % to about 1.4 wt %, from about1 wt % to about 1.4%, or from about 1.1 wt % to about 1.3 wt %) perkilogram of the egg white. Conventionally, efforts to create ahomogeneous, low viscosity egg white suitable for bulk chromatographicprocessing requires adding an acidic buffer having a volume 2- to 5-fold(i.e., 200% to 500%) of the volume of the egg white as it was notexpected that adding a small amount of an acidic buffer would beeffective in adjusting the pH of the egg white to the target value. Sucha process is not economically practical or feasible at a largemanufacturing scale due to the size constraints of chromatographiccolumns and other manufacturing equipment used in the egg whitepreparation process. Unexpectedly, the present inventors discovered thata homogeneous, low viscosity egg white suitable for bulk chromatographicprocessing can be obtained by adding a small amount of an acidic buffer(e.g., at most about 5 wt % per kilogram of egg white) relative to theamount of egg white, thereby significantly reducing the amount of thecolumns used in the chromatographic isolation processes, the sizes ofother equipment used in these processes, and the cost and time ofisolating recombinant proteins from egg white.

Without wishing to be bound by theory, it is believed that additionaladvantages of adding a relatively smaller amount of an acidic bufferrelative to the amount of egg white include (1) negligible dilution ofegg white, (2) essentially no change in conductivity, which allows forthe precipitation of ovomucin-lysozyme complexes from egg white, whichin turn reduces the viscosity of the egg white at lower pH range (e.g.,pH 5-6.5) and facilitates separation of unwanted materials from the eggwhite during the filtration and chromatographic processes, and (3)minimizing formation of low pH pockets within the viscous egg white thatcan potentially damage recombinant proteins or result in uneven pH ofthe egg white materials.

In general, the acidic buffer can have a conductivity ranging from about8 mS/cm to about 40 mS/cm. For example, the acidic buffer can have aconductivity of at least about 8 mS/cm (e.g., at least about 9 mS/cm, atleast about 10 mS/cm, at least about 11 mS/cm, at least about 12 mS/cm,at least about 13 mS/cm, at least about 14 mS/cm, at least about 15mS/cm, at least about 16 mS/cm, at least about 17 mS/cm, at least about18 mS/cm, at least about 19 mS/cm, at least about 20 mS/cm, at leastabout 21 mS/cm, at least about 22 mS/cm, at least about 23 mS/cm, atleast about 24 mS/cm, at least about 25 mS/cm) and/or at most about 40mS/cm (e.g., at most about 39 mS/cm, at most about 38 mS/cm, at mostabout 37 mS/cm, at most about 36 mS/cm, at most about 35 mS/cm, at mostabout 34 mS/cm, at most about 33 mS/cm, at most about 32 mS/cm, at mostabout 31 mS/cm, at most about 30 mS/cm, at most about 29 mS/cm, at mostabout 28 mS/cm, at most about 27 mS/cm, at most about 26 mS/cm, or atmost about 25 mS/cm). For example, the acidic buffer can have aconductivity of any value between about 8 mS/cm and about 40 mS/cm.Without wishing to be bound by theory, it is believed that using anacidic buffer having a conductivity from about 8 mS/cm to about 40 mS/cmwould minimize the changes to the conductivity of the mixed egg white sothat the process can be implemented and streamlined with the downstreamprotein isolation step without changing the isolation conditions of thecolumn chromatography used in the isolation step and without causingfurther precipitation or aggregation.

In some embodiments, the acidic buffer can be added to the egg whitepool in a single bolus injection. In some embodiment, the single bolusinjection is performed at a suitable injection rate (e.g., at leastabout 1 L/minute) to ensure that the addition of the acidic buffer isadded within a suitable amount of time (e.g., at most about 5 minutes).Without wishing to the bound by theory, it is believed that theadvantages of using a single bolus injection include (1) formingrelatively large flocculent that settles easily under gravity, therebyreducing the need for large filters and (2) avoiding the need forcontinuous titration of a viscous egg white pool, which can cause falsepH readings that trigger repeated addition of an acid or a base, whichin turn can potentially damage the recombinant proteins in the egg whiteand increase the turbidity of the egg white solution produced.

In some embodiments, after the acidic buffer is added to the egg whitepool, the egg white is mixed at a suitable temperature (e.g., from about2° C. to about 25° C.) for a suitable period of time (e.g., at leastabout 1 hour) to form a mixed egg white having a suitable pH. In someembodiments, the addition of the acidic buffer and the mixing of the eggwhite are performed concurrently.

In general, the addition of the acidic buffer and the mixing of theacidic buffer with the egg white result in formation of a large amountof precipitates (e.g., ovomucin-lysozyme complexes). The precipitatesgenerally reduce the viscosity of the egg white remaining in thesolution.

In some embodiments, the pH of the mixed egg white is a value such thatthe mixed egg white is rendered least viscous. For example, the mixedegg white can have a pH at least about 5 (e.g., at least about 5.2, atleast about 5.4, at least about 5.6, at least about 5.7, at least about5.8 or at least about 5.9) and/or at most about 6.5 (e.g., at most about6.3, at most about 6.2 or at most about 6.1). In some embodiments, themixed egg white can have a pH of about 6. Without wishing to be bound bytheory, it is believed that such a pH (e.g., about 6) can allowformation of the largest amount of precipitates from egg white thatsettle under gravity, thereby reducing the need for filtration andfacilitating formation of a homogeneous, low viscosity egg whitesolution.

In general, the mixed egg white (i.e., the acidified or pH adjusted eggwhite) has a relative low conductivity (e.g., similar to theconductivity of an egg white not treated with an acidic buffer). Forexample, the mixed egg white can have a conductivity of at least about 8mS/cm (e.g., at least about 8.2 mS/cm, at least about 8.4 mS/cm, atleast about 8.6 mS/cm, at least about 8.8 mS/cm, at least about 9 mS/cm,at least about 9.2 mS/cm, at least about 9.4 mS/cm, at least about 9.6mS/cm, at least about 9.8 mS/cm, at least about 10 mS/cm, at least about11 mS/cm, at least about 12 mS/cm, at least about 13 mS/cm, or at leastabout 14 mS/cm) and/or at most about 20 mS/cm (e.g., at most about 19mS/cm, at most about 18 mS/cm, at most about 17 mS/cm, at most about 16mS/cm, at most about 15 mS/cm, at most about 14 mS/cm, at most about 13mS/cm, at most about 12 mS/cm, at most about 11.8 mS/cm, at most about11.6 mS/cm, at most about 11.4 mS/cm, at most about 11.2 mS/cm, at mostabout 11 mS/cm, at most about 10.8 mS/cm, at most about 10.6 mS/cm, atmost about 10.4 mS/cm, at most about 10.2 mS/cm, or at most about 10mS/cm). For example, the mixed egg white can have a conductivity of anyvalue between about 8 mS/cm and about 20 mS/cm. Without wishing to bebound by theory, it is believed that keeping the mixed egg white atconductivity from about 8 mS/cm to about 20 mS/cm would allow the mixedegg white to comply with the conditions used in the downstream proteinisolation step so that the mixed egg white can be consistently used inthe isolation step without changing the isolation conditions of thecolumn chromatography used in this step.

After the mixing is completed, the methods described herein can includean optional step of allowing the mixed egg white to settle for asuitable period of time (e.g., at least about 6 hours) such that the eggwhite separates into top, middle and bottom layers. Typically, the toplayer includes certain unwanted materials (e.g., denatured protein andfoamy lipids including phospholipids, triglyceride, and cholesterol)having a low density, the bottom layer includes the precipitates formedfrom the egg white proteins (e.g., ovomucin-lysozyme complexes), and themiddle layer includes a relatively clear egg white solution.

In some embodiments, during the settling step, if the pH of the mixedegg white becomes falling outside the desired value (e.g., 5.7 ±0.1, 6.0±0.1, or 6.3 ±0.1), it can be adjusted to reach the desired value byusing an acid (e.g., the acidic buffer described above) or a base (e.g.,a 5.7 M sodium acetate or 1 N sodium hydroxide solution). In suchembodiments, the pH adjustment can be followed by additional mixing(e.g., for at least about 1 hour) and settling (e.g., for at least about3 hours) at room temperature. In general, the total mixing and settlingtime does not exceed 24 hours to minimize the time that the exogenousproteins in the egg white are exposed to the processing environment,thereby maintaining the biological activities of these proteins.

In some embodiments, the methods described herein can include a step ofisolating the middle layer from the mixed egg white after the settingstep. In general, the middle layer can be isolated by using methodsknown in the art. For example, the middle layer can be siphoned out ofthe vessel containing the mixed egg white by inserting a tube (e.g., ametal or polycarbonate tube) into the middle layer (preferably in thecenter of the middle layer) so that the content of the middle layer canbe pumped into a receiving vessel without disturbing the top and bottomlayers.

In general, after the middle layer is isolated, at least a portion ofthe middle layer (e.g., all of the middle layer) can be filtered toremove any particles suspended in the middle layer to obtain ahomogeneous, low viscosity, clear egg white solution. This step is alsoknown as egg white clarification. In some embodiments, the middle layercan be filtered through one or more filters having an average pore sizeat most about 100 μm (e.g., at most about 90 μm, at most about 80 μm, atmost about 70 μm, at most about 60 μm, at most about 50 μm, at mostabout 40 μm, at most about 30 μm, at most about 20 μm, at most about 10μm, at most about 9 μm, at most about 8 μm, at most about 7 μm, at mostabout 6 μm, at most about 5 μm, at most about 4 μm, at most about 3 μm,at most about 2 μm, or at most about 1 μm) and/or at least about 0.1 μm(e.g., at least about 0.2 μm, at least about 0.3 μm, at least about 0.4μm, at least about 0.5 μm, at least about 0.6 μm, at least about 0.7 μm,at least about 0.8 μm, at least about 0.9 μm, at least about 1 μm, atleast about 2 μm, or at least about 3 μm). For example, the filter canhave an average pore size ranging from about 0.1 μm to about 100 μm(e.g., from about 0.1 μm to about 40 μm, from about 40 μm to about 100μm, from about 3 μm to about 6 μm, or from about 0.1 μm to about 0.3μm).

In some embodiments, the middle layer can be filtered through aplurality of filters serially connected to each other, in which theaverage pore size of the filters decreases sequentially. For example,the middle layer can be filtered through a filtration system containingthree serially connected filters, in which the first filter can have anaverage pore size of about 40 μm, the second filter can have an averagepore size from about 3 μm to about 6 μm, and the third filter can havean average pore size from about 0.1 μm to about 0.3 μm. An example ofsuch a filtration system is a system containing sequential depth filterscommercially available from Pall Corporation (e.g., containing T2600,K200P and Bio 10 depth filters). Optionally, the filtration system canfurther include a fourth filter (e.g., having an average pore size about0.2 μm) downstream of the third filter. An example of the fourth filteris a Sartobran P filter available from Sartorius Corporation.

In some embodiments, the filters used to filter the middle layer canhave a large filtration medium area. For example, the filters can have afiltration medium area of at least about 1 m² (e.g., at least about 2m², at least about 4 m², at least about 6 m², or at least about 8 m₂.

In some embodiments, after the acidic buffer and the egg white are mixedto form a mixed egg white, the mixed egg white can be filtered withoutthe need to allow the precipitates in the mixed egg white to settle. Insuch embodiments, the mixed egg white (including the precipitates formedduring the addition/mixing steps and the remaining egg white solution)can be filtered without any prior separation of the precipitates fromthe mixed egg white. For example, after the acidic buffer and the eggwhite are mixed, the mixed egg white can be filtered without settling byusing one or more filters described herein (e.g., a filtration systemcontaining three serially connected filters having decreasing poresizes). Without wishing to be bound by theory, it is believed that, byeliminating a settling step, such a method can significantly reduce thetime and costs for preparing egg white for bulk chromatographicprocessing and for isolating proteins from the egg white.

In some embodiments, after the acidic buffer and the egg white are mixedto form a mixed egg white, the mixed egg white can be centrifuged toseparate precipitates (e.g., ovomucin-lysozyme complexes) from thesupernatant. The supernatant thus obtained can then be filtered by usingone or more filters described herein.

In general, the filtered egg white solution can be used to isolaterecombinant proteins by using column chromatography, such as ionexchange chromatography or chromatography based on hydrophobicinteraction. Examples of such chromatographic methods have beendescribed, e.g., in U.S. Application Publication No. 2009/0299037.

In some embodiments, this disclosure features methods of isolating arecombinant protein from egg white. For example, such methods caninclude the steps of: (1) providing a pool of egg white containing arecombinant protein, the pool having a volume of at least about 10liters; (2) adjusting the pH of the egg white to from about 5 to about6.5, wherein the conductivity of the pH-adjusted egg white is from about8 mS/cm to about 20 mS/cm; (3) filtering the egg white to form asolution; and (4) isolating the recombinant protein in the egg white bycolumn chromatography. The adjusting step can be performed by adding asmall amount of an acidic buffer (e.g., from about 0.5 wt % to about 5wt % per kilogram of the egg white) to the egg white in the same manneras described above. The filtering and isolating steps can be performedby the methods described herein or methods known in the art.

In some embodiments, this disclosure features methods of filteringacidified egg white (e.g., egg white acidified by an acidic bufferdescribed above). For example, such methods can include the steps of:(1) passing a pre -treatment buffer having a conductivity between about8 mS/cm and about 20 mS/cm through a filter; and (2) passing egg white(e.g., having a volume of at least about 50 L) having a pH from about 5to about 6.5 through the filter to obtain a filtered egg white. Thefiltered egg white can then be used to isolate recombinant proteins byusing column chromatography. In some embodiments, the filter used insuch methods can be similar to or the same as those described above. Forexample, the filter can have the same average pore size (e.g., fromabout 0.1 μm to about 100 μm) or filtration medium area as thosedescribed above (e.g., at least about 8 m²).

In some embodiments, the pre-treatment buffer can be used to wet thefilter prior to passing the egg white (e.g., acidified egg white). Thepre-treatment buffer can include one or more salts (e.g., alkalinesalts). Exemplary salts include sodium phosphate and sodium chloride. Insome embodiments, the pre-treatment buffer can include a combination ofsodium phosphate and sodium chloride.

In general, the pre-treatment buffer can include an acid. Exemplaryacids include acetic acid, hydrochloric acid, sulfuric acid, nitricacid, phosphoric acid, hydrofluoric acid, hydrobromic acid, perchloricacid, citric acid, boric acid, tartaric acid, lactic acid, formic acid,oxalic acid, uric acid, and barbituric acid. In some embodiments, acombination of two or more (e.g., three or four) acids can be used inthe pre-treatment buffer.

In some embodiments, the pre-treatment buffer can have a pHsubstantially the same as the pH of the egg white. For example, thepre-treatment buffer can have a pH at least about 5 (e.g., at leastabout 5.2, at least about 5.4, at least about 5.6, at least about 5.7,at least about 5.8 or at least about 5.9) and/or at most about 6.5(e.g., at most about 6.4, at most about 6.3, at most about 6.2 or atmost about 6.1). In some embodiments, the pre-treatment buffer can havea pH of about 6. Without wishing to be bound by theory, it is believedthat using a pre-treatment buffer having a pH substantially the same asthe pH of the egg white to treat a filter can adjust the pH of thefilter surface to be similar to the pH of the egg white, therebyminimizing precipitation of the egg white during filtration, which cancause blockage in the filter or obstruct the flow of the samples thatcauses shear stress on the filter, thereby seriously shortening theusage life of the filter.

Generally, the pre-treatment buffer can have a conductivity compatiblewith the conductivity of the egg white such that the filtered acidifiedegg white is compatible with the characteristics of the columnchromatography (e.g., ion exchange chromatography) used in thedownstream isolation/purification processes. For example, thepre-treatment buffer can have a conductivity of at least about 8 mS/cm(e.g., at least about 9 mS/cm, at least about 10 mS/cm, at least about11 mS/cm, at least about 12 mS/cm, at least about 13 mS/cm, at leastabout 14 mS/cm, at least about 15 mS/cm, at least about 16 mS/cm, atleast about 17 mS/cm, at least about 18 mS/cm) and/or at most about 20mS/cm (e.g., at most about 19 mS/cm, at most about 18 mS/cm, at mostabout 17 mS/cm, at most about 16 mS/cm, at most about 15 mS/cm, at mostabout 14 mS/cm, at most about 13 mS/cm, at most about 12 mS/cm, or atmost about 11 mS/cm). For example, the pre-treatment buffer can have aconductivity of any value between about 8 mS/cm and about 20 mS/cm. Thepresent inventors found that using a filter without being treated withthe above pre-treatment buffer to filter an egg white solution wouldcause the filter to be clogged rapidly by the precipitates formed duringthe filtration process. On the other hand, the present inventors foundunexpectedly that using a pre-treatment buffer having the aboveconductivity to treat a filter can adjust the conductivity of the filtersurface to be similar to that of the egg white, thereby minimizingprecipitation of the egg white during filtration and significantlyincreasing the life time of the filter.

In some embodiments, the egg white can be passed through a filter undera relatively small differential pressure (e.g., less than about 30 psi,less than about 25 psi, less than about 20 psi, less than about 15 psi,less than about 12 psi, less than about 10 psi, or less than about 5psi). Without wishing to be bound by theory, it is believed that passingthe egg white through a filter under a relatively small differentialpressure can minimize damages and/or blockage to the filter, therebyreducing product costs as the filter can be very expensive.

The contents of all publications cited herein (e.g., patents, patentapplication publications, and articles) are hereby incorporated byreference in their entirety.

The following examples are illustrative and not intended to be limiting.

Example 1 : Method of Preparing Egg White for Bulk ChromatographicProcessing Having a Settling Step

Fifty to four hundred kilograms of frozen egg white stored at −20° C. in4L Nalgene bottles was thawed at room temperature in water baths set to21±1° C. for approximately 5-7 hours. Once every hour, each bottle wasremoved from the water bath, visually inspected to determine degree ofthaw, inverted repeatedly and placed back into the water bath until thethaw was complete. Thawed bottles were removed from the water baths andstored at 2-8° C. After the last egg white bottle was thawed, the eggwhite in the bottles was pooled into an open top mixing vessel. Anacidic buffer containing 5.7 M sodium acetate at pH 4.0 (about 1.3%wt/wt with respect to the weight of the egg white) was added to thethawed egg white pool at 1 kilogram per minute, ensuring that theduration of the addition of the acidic buffer did not exceed 5 minutes,to obtain a final target pH of 6.0±0.1 at 2-8° C. The egg white mixturewas stirred continuously for 1 hour in the open top mixing vesselwithout temperature control, and then decanted into a closed single usemixer, refrigerated at 2-8 ° C., and mixed for 6 hours. After the mixingwas stopped, the precipitate was allowed to settle for 6 hours. Ifrequired, the pH was further adjusted to 6.0±0.1 at 2-8° C. using either5.7 M sodium acetate or 1 N sodium hydroxide, followed by additionalmixing for 1 hour and settling for 3 hours at room temperature (totalmixing and settling time not to exceed 24 hours). After the settling iscompleted, the mixed egg white formed three layers, i.e., top, middle,and bottom layers. A tube was then placed into the middle layer and themiddle layer was siphoned or pumped out through the tube withoutdisturbing the top and bottom layers. Filters (sequential PallCorporation depth filters, 40 micron, 3-6 micron, 0.1-0.3 micron,respectively) were pre-rinsed with 80 liters of purified water per metersquared of filter area to remove total organic carbon, leachables, andextractables, and then drained. The pre-rinsed filters were then treatedwith 1 filter hold up volume of a pre -treatment buffer solutioncontaining 20 mM sodium phosphate and 140 mM sodium chloride at pH 6.0and then drained. The egg white solution was decanted, avoiding thesettled precipitated and aggregated egg white particulates, and filteredthrough dead end filtration at a flow rate of 1 liter per meter squaredper filter type or a differential pressure less than 30 psid to removeany un-settled precipitated material and collected into a sterile singleuse mixer. At the completion of egg white filtration, the retained eggwhite in the filtration system was flushed with one filter train hold upvolume of the pre-treatment buffer solution containing 20 mM sodiumphosphate and 140 mM sodium chloride at pH 6.0 to recover product andcollected in a sterile single use mixer. The filtered egg white solutionwas sampled for measurement of enzyme activity and ultraviolet (UV)absorbance and stored at 2-8° C. for up to 24 hours before it was usedfor isolation of recombinant proteins in the egg white via columnchromatography. Table 1 shows the results of egg white acidification byusing the acidic buffer described above (i.e., 5.7 M NaOAc, pH 4.0, and1.3% wt/wt).

TABLE 1 Average Total Weight (kg) of the Mixed 83.52 ± 2.03  Egg WhiteNative pH 8.41 ± 0.06 Native Conductivity (mS/cm) 8.47 ± 0.25 5.7MNaOAc, pH: 4.0 (mL)   1 ± 0.02 %5.7M NaoAC added (w/w) 1.30% %5.7M NaOAcadded (v/w) 1.20% Time Post Bolus Conductivity Addition (min) pH (mS/cm)10 5.79 ± 0.03 9.52 ± 0.08 30 5.92 ± 0.02 9.45 ± 0.08 90 5.93 ± 0   9.48± 0.09 180 6.01 ± 0.01 9.48 ± 0.06 1080 6.09 ± 0.01 9.49 ± 0.09

Example 2: Method of Preparing Egg White for Bulk ChromatographicProcessing Without a Settling Step

Four hundred kilograms of frozen egg white (+/−10%>) of frozen egg white(−20° C.) was thawed at 2-8° C. (in a walk-in cold room) inapproximately 24-72 hours. The thawed egg white was pooled into ajacketed single use closed mixing vessel and maintained at 2-8° C. Anacidic buffer containing 5.7 M sodium acetate at pH 4.0 (1.08% wt/wt)was added to the thawed egg white at 1 kilogram per minute, ensuringthat the duration of the addition of the acidic buffer did not exceed 5minutes, to obtain a final target pH of 6.0±0.5. The acidified egg whitesolution was stirred continuously for 3 hours at 2-8° C. The acidifiedegg white was then warmed to 21±3° C. via the jacketed tank prior tofiltration. Filters (sequential Pall Corporation depth filters, 40micron, 3-6 micron, 0.1-0.3 micron, respectively) were pre-rinsed with80 liters of purified water per meter squared of filter area to removetotal organic carbon, leachables, and extractables. The purified waterin the filter train was displaced with a pre-treatment buffer solutioncontaining 20 mM sodium phosphate and 140 mM sodium chloride at pH 6.0until the filter effluent conductivity was within the acceptableconductivity range of the pre-treatment buffer (e.g., 10 - 15 mS/cm).The homogeneously mixed acidified egg white was filtered through deadend filtration at a flow rate of 1 liter/min/m² per filter type orresulting in a differential pressure≤10 psi to remove any precipitatedor aggregated material. The initial filter effluent volume equal to 80%of the filter train hold up volume was used to displace thepre-treatment buffer and was discarded prior to product collection. Theremaining filtered pre-treatment buffer and egg white was collected intoa sterile single use mixer. At the completion of egg white filtration,the retained egg white in the filtration system was flushed with 1.5filter train hold up volumes (0.5 holdup volumes are collected while 1hold-up volume remains in the filtration train) of the pretreatmentbuffer solution containing 20 mM sodium phosphate and 140 mM sodiumchloride at pH 6.0 to recover product and collected in the sterilesingle use mixing tank. The filtered egg white was sampled formeasurement of enzyme activity and absorbance and stored at 2-8° C. forup to 24 hours before it is used for isolation of recombinant proteinsin the egg white via column chromatography.

Example 3: Scale Down of Egg White Manufacturing Process to EvaluateDirect Loading on Depth Filtration Performance and Robustness of EggWhite Acidification Materials

All chemicals used for the Clarification studies were of USP/MC grade.Egg white source materials are listed in Table 2. All source materialwas stored at -20° C. and thawed at 2-8° C. 48-72 hours prior to use.Source material was pooled and maintained at 2-8° C. up through theacidification step.

TABLE 2 Egg White Source Materials Egg White Titer Run Aliquot (L)(g/L)* Zygosity 1 20 0.77 Homo 2 20 1.02 Homo 3 20 0.79 Homo *Based onenzymatic activity

For all buffer preparations, conductivity measurements were conductedutilizing pH/conductivity meters with temperature compensated to 25° C.pH of buffers were measured at 20° C. In-process buffer preparationformulations are listed in Table 3 below.

TABLE 3 Buffer Formulation Specifications Cond Buffer Raw Material g/LpH (mS/cm) Egg White 5.7M Sodium Sodium Acetate 136.1 4.0 ± 0.1 36.0 ±5.0 Acidification Acetate, pH 4.0 Trihydrate Glacial Acetic Acid 285.0Filter Flush/ 20 mM Sodium NaH₂PO₄•H₂O 2.24 6.0 ± 0.1 16.0 ± 2.0Equilibration Phosphate, 140 mM Na₂HPO₄•7H₂O 1.02 NaC1, pH 6.0) NaCl8.18 Wash 1 5 mM Sodium NaH₂PO₄•H₂O 2.24 6.0 ± 0.1  0.45 ± 0.15Phosphate, pH 6.0 Na₂HPO₄•7H₂O 1.02 Wash 2 5 mM Tris, 1M Tris Base 0.617.2 ± 0.1 88.0 ± 5.0 NaC1, pH 7.2 NaC1 58.44 Wash 3 5 mM Tris, 0.25MTris Base 0.61 7.2 ± 0.1 23.0 ± 3.0 NaC1, pH 7.2 NaC1 14.61 Elution 5 mMTris, 17% Tris Base 0.61 7.2 ± 0.1 <2.0 IPA, pH 7.2 Isopropyl Alcohol133.0 Acid 0.85% Phosphoric Phosphoric Acid 16.9 N/A N/A Cleaning Acid

Process Equipment

Process skids (AKTA Explorers) were serviced (preventive maintenance) byGE healthcare. Process equipment (Pall Stax chassis and AKTA Explorers)was maintained by Synageva PD personnel during the entirecharacterization study. Table 4 below lists all hardware equipment usedin this Example.

TABLE 4 Equipment List Equipment Step Description Manufacture Part No.General Accumet XL550 Fisher X13-12005 pH/conductivity Scientific meter3.2 kg Bench Scale Mettler Toledo ML3002E 50 kg Floor Scale Ohaus CD-33Masterflex L/S Pump Cole-Parmer 7523-60 (10-600 rpm) Masterflex I/P 73Cole Parmer 06427-73 C-flex Masterflex L/S 24 Cole Parmer 06508-24Bioprene Clarification Stax Pilot Chassis Pall SXLSC02W Stax PilotChassis Pall SXLSC02W Masterflex I/P Pump Cole-Parmer 77410-10 (33-600rpm) Pressure Flow Cell SciLog 080-696-PSX-5 SciPres Pressure SciLog080-690 Monitor 250 kg Floor Scale Ohaus CD-33 PHIC SDM AKTA Explorer GEHealthcare 29001622 XK16 Column GE Healthcare N/A A280 Nanodrop 2000Thermo N/A Scientific SDS-PAGE Mini-PROTEAN Bio-Rad 456-1096 TGX 4%-20%(L/N: 400091244) Precision Plus Dual Bio-Rad 161-0374 Color Standards(L/N: 35001785) Mini PROTEAN Bio-Rad 165-8000 Tetra Cell

Pall STAX chassis (PN# SXLSC02W) were set up using the STAX depthfilters listed in Table 5 in the following configuration (40 μm, 3-6 μm,and 0.1-0.3 μm) and an additional Sartobran P 0.2 μm filter.

Chassis #1 : (bottom) Manifold→T2600→Vent plate (top)

Chassis #2: (bottom) Manifold→K200P→Vent plate→Manifold→Bio 10→Ventplate (top)

Each filter was sandwiched in between a 1.5″ inlet/outlet manifold (P/N:7008225) and a top Vent plate. Hold up volume of each individual filterwas determined empirically during the initial water flush.

TABLE 5 Depth Filtration Equipment Load Pore Stax Hold Up Ratio SizeFilters Vol Run Filter (L/m²) (um) (S.A. m²) (L) 1 T2600 20 40 1.0 6.5K200P 20 3-6 1.0 6.0 BIO10 20 0.1-0.3 1.0 5.5 Sartobran P 160 0.22 1.81.35 2 T2600 20 40 1.0 6.5 K200P 20 3-6 1.0 6.0 BIO10 40 0.1-0.3 0.5 3.8Sartobran P 160 0.22 1.8 1.35 3 T2600 20 40 1.0 6.5 K200P 20 3-6 1.0 6.0BIO10 40 0.1-0.3 0.5 4.0 Sartobran P 160 0.22 1.8 1.35

Columns XK 16/20 (GE Healthcare) were used for all phenyl hydrophobicinteraction chromatography (PHIC) column packing. All chromatography wasperformed on an AKTA Explorer equipped with Unicorn software version5.31 (GE Healthcare).

Procedures

Before starting the clarification step, frozen egg white aliquots(totaling 20 L) were thawed for 2-8° C. for 48-72 hours. The thawed eggwhite was pooled into an appropriately sized polypropylene tank (25 L)with sterile media liner (Thermo Scientific/Hyclone P/N 343050-0005).The pooled egg white was mixed to homogeneity using an overhead mixer(330 rpm) at 2-8° C. The pooled egg white was then conditioned to thetarget pH through addition of 5.7 M sodium acetate at pH 4.0. Table 6lists the volume of the 5.7 M sodium acetate added for eachclarification run to achieve the target pH. The pH was monitored formore than 2 hours to ensure target pH was achieved.

TABLE 6 Pooled Egg White Acidification 5.7M 5.7M 5.7M Pool TargetAcetate Acetate Acetate Initial Final Acidification Run Vol. (L) pH Vol.(mL) Vol. (% wt/wt) Vol. (% v/v) pH pH Time (h) 1 20 6.0 180 0.94 0.90N/A 5.94 18 2 20 5.7 245 1.27 1.22 8.11 5.67 24 3 20 6.3 164 0.83 0.808.21 6.25 6

Prior to filtration, filters were flushed individually with 80 L/m² withfiltered RO/DI water. During the water flush, the holdup volume for eachfiltered was empirically determined. Upon completion of the water flush,the filters were plumbed into a continuous train and flushed with 16L/m² of PHIC equilibration buffer (20 mM sodium phosphate, 140 mM sodiumchloride, pH 6.0). Buffer flush ended when pH and conductivity of theeffluent met flush buffer specifications (e.g., pH 6.0±0.1, conductivity16.0±2.0). Pooled egg white was loaded onto the filter train using a ⅜″dip tube (I/P 73 tubing Cole) at 1.0 L/min with continuous mixing(overhead mixer, 300 rpm). 80% of the measured, cumulative hold upvolume (˜13.2 L) was collected and directed to waste. Filtrate was thencollected in a separate appropriately sized container until thecongealed low density precipitate entered dip tube. Filtration train wasthen flushed with 1.5 hold up volumes (˜25 L) with a bufferequilibration buffer. The final filtrate was mixed well using a cleantank paddle to ensure homogeneity prior to sampling. Filtrate was storedat 2-8° C. overnight prior to PHIC separation.

Prior to PHIC separation, filtrate stored at 2-8° C. was warmed using aroom temperature water bath. The filtrate was secondarily filteredthrough a Sartobran 150 cartridge (0.45um/0.22um, P/N 5231307H4-00) togenerate the final PHIC load. Table 7 lists the buffers and in-processparameters used.

TABLE 7 PHIC Chromatography Unit Operation Steps Linear Flow VelocityRate Step Buffer CV (cm/h) (mL/min) Equilibration 20 mM SodiumPhosphate, 5 120 cm/h 4.02 140 mM NaCl, pH 6.0 Loading  60 cm/h 2.01Wash 1 5 mM Sodium Phosphate, 8 120 cm/h 2.01 (1CV) pH 6.0 4.02 (7CV)Wash 2 5 mM Tris, 1M NaCl, 8 120 cm/h 4.02 pH 7.2 Wash 3 5 mM Tris,0.25M NaCl, 4 120 cm/h 4.02 pH 7.2 Pre-Elution 5 mM Tris, 17% IPA, pH7.2 UV→40 mAU 120 cm/h 4.02 Fraction 1 5 mM Tris, 17% IPA, pH 7.2 UV→800mAU  120 cm/h 4.02 Elution 5 mM Tris, 17% IPA, pH 7.2 1.9 120 cm/h 4.02Post Elution 5 mM Tris, 17% IPA, pH 7.2 2.0 120 cm/h 4.02 Strip RO/DI 560 cm/h 2.01 Upflow Acid 0.85% Phosphoric Acid 3 60 cm/h 2.01 CleaningUpflow Water Rinse RO/DI 5 60 cm/h 2.01 Upflow CIP 0.5N NaOH 3 60 cm/h2.01 Upflow (1 h hold) Water Rinse RO/DI 5 60 cm/h 2.01 Upflow Storage20% Ethanol 3 60 cm/h 2.01 Upflow

Results and Discussion

(1) Direct loading of acidified egg white (i.e., no settling step)

Initially, a single center point run (“Run 1”; 1 :20 scale; 20 Lacidified egg white at pH of about 6) was conducted to evaluate theimpact of direct loading (no settling, loading ration of 20 L/m²) ondepth filtration performance in order to establish a representativeexperimental scale model. For each filter (i.e., T2600, K200P, Bio 10),although the inlet feed pressure in the filter did increase linearlyover the course of acidified egg white loading, the differentialpressure did not exceed 10 psid. In addition, feed pressure did notincrease further during the buffer flush and exhibited a dramaticdecrease in filter T2600. These results suggest that direct loading ofthe acidified egg white to the filters did not significantly block thefilters during this run.

The protein recovery results are summarized in Table 8 below. As shownin Table 8, protein recovery after the clarification step met targetexpectation (>70%).

TABLE 8 Protein Recovery After Clarification Ave. Adj Recovered resultsVolume Protein Run 1 (U/mL) (mL) (mg) Egg White Pool 199.5 20000 15344.6Acidified Egg White Pool 253.4 20000 19493.6 Clarified Egg White Pool122.8 32700 15438.2 Clarif Step Yield* 100.6% (Thawed Egg White) ClarifStep Yield* 79.2% (Acidified Egg White) *Based on Enzymatic Activity

PHIC column performance of Run 1 was comparable to the results obtainedfrom runs including a settling step of the acidified egg white. Inaddition, protein purity was measured by a SDS-PAGE analysis using 4-20%Tris-Glycine gel. PHIC eluent fractions from Run 1 did not reveal anydifferences in banding pattern when compared to runs including asettling step. These data suggested that direct loading of acidified eggwhite during clarification (i.e., filtration) did not impact proteinrecovery or PHIC performance in terms of yield and purity.

The above results suggest that acidified egg white can be directlyloaded to the filters during the clarification step without goingthrough a settling step. This method was then applied to the Robustnessstudy runs (i.e., Runs 2 and 3) described in the next section.

(2) Robustness of egg white acidification

A 2-factor, 2-level experimental design (high/low, low/high) was used toevaluate the robustness of the egg white acidification step. Theexperimental conditions are defined in Table 6. In Run 2, acidificationwas performed for 24 hours to achieve a final pH of 5.67 (i.e., about5.7). In Run 3, acidification was performed for 6 hours to achieve afinal pH of 6.25 (i.e., about 6.3). The results show that both runsexhibited comparable linear feed pressure increases and maximum feedpressures due to increased T2600 fouling. A lower maximum feed pressurewas observed in Run 2, which is believed to be due to a correlationbetween egg white pH and egg white viscosity (i.e., lower egg white pHresulting in lower egg white viscosity). In no case did the feedpressure exceed 10 psig, showing comparable performance to Run 1.

After the clarification step, the protein recoveries in Runs 2 and 3respectively were 71% and 83%, which met target expectations of(i.e., >70%>) and were comparable to Run 1 (i.e., 79%). After PHICseparation, the protein yields in Runs 2 and 3 respectively were 59% and68%, which were comparable to those obtained from runs including asettling step. Chromatogram overlays comparing Runs 2 and 3 to both Run1 confirmed comparable column performance.

The above results suggest that variation of acidification pH and timedid not result in any significant negative impact relative to enzymeactivity or yield. Thus, the clarification step was robust whenconducted within two tested ranges (i.e., acidification pH 5.7-6.3 andacidification time of 6-24 hours).

(3) Egg white in-process stability

Process hold studies were conducted to define maximum hold times for thefollowing three unit operation hold points: (1) thawed egg white (2-8°C.), (2) acidified egg white (2-8° C. and ambient temperature), and (3)clarified egg white (2-8° C. and ambient temperature). Stability of theproduct (in terms of enzymatic activity) was assessed over a 72 - 96hour time period. Two separate stability studies were conducted toassess impact on clarification parameter variance on product hold timesat the acidified and clarified egg white steps. Sixty mL aliquots (twofor each of acidified and clarified egg white) were taken from each ofthe two robustness experiments above (i.e., Runs 2 and 3) and stored ateither 2-8° C. or ambient temperature. 1.5 mL samples were taken every24 hours up to either 72 hours (pH 5.7/24 hours) or 96 hours (pH 6.3/6hours). Due to time constraints, a stability study was not conducted onRun 1 (center point). The stability design parameters are summarized inTable 9 below.

TABLE 9 In Process Stability Design Study Parameters CommercialCommercial Study Process Target Hold Temp Temp Acidification Hold HoldPoint (hr) (° C.) (° C.) pH Time (h) Time (h) Thawed egg 72-96 2-8 2-8N/A N/A 0-168* white 2-8 N/A N/A Acidified 6 hr 2-8 2-8 5.7-6.3 6-240-72 (24 h) egg white (6-24 hr range) RT** 0-96 (6 h)  Clarified ≤6 hrRT or 2-8 2-8 5.7-6.3 6-24 0-72 (24 h) egg white (no refiltration) RT**0-96 (6 h)  ≤24 hr (with refiltration) *T = 0 equals the beginning ofthe Thaw (additional hold time including thaw time) **RT = 18-25° C.(Average: 22° C.)

The results show that enzymatic activity for all three in-process holdpoints (thawed, acidified, and clarified egg white) was stable over theentirety of the time course study (up to 96 hours) at 2-8° C. Inaddition, variation of either the acidification pH or acidification timein general did not negatively impact enzymatic activity. A single set oftest parameters (pH 5.7, room temperature) for the acidified egg whitehold point did exhibit a ˜20% decrease in enzymatic activity. However,the current commercial process storage target for the acidified eggwhite is 2-8° C. and, therefore, this observation is not a risk to thecommercial process. Based on the data, the thawed/pooled egg white wasstable for up to 168 hours (including the initial 72 hour thaw time) at2-8° C., the acidified egg white was stable up to 96 hours at 2-8° C.,and the clarified egg white stage was stable up to 96 hours at 2-8° C.or room temperature.

(4) Supplemental DNA clearance study

Although egg white itself does not contain DNA, the egg white harvestingprocess may result in the presence of host genomic DNA through theintroduction of minor amounts egg yolk into the egg white pool.Acidified and clarified egg white derived from the studies of Runs 1-3above were analyzed for host genomic DNA. The analyses revealed thefollowing:

1. DNA was detected in the pooled and acidified egg white

2. Significant DNA clearance during the Clarification step wasidentified.

These data suggested that in addition to egg white host proteinclearance, the clarification step provides a means to remove hostgenomic DNA from the product pool.

Other embodiments are within the scope of the following claims.

1-50. (canceled)
 51. A method of isolating a recombinant protein fromegg white comprising the steps of: providing a pool of egg whitecomprising the recombinant protein; wherein the recombinant protein isexogenous to egg white; adding an acidic buffer comprising an acidicagent to the pool of egg white, the acidic buffer being from about 0.5wt % to about 5 wt % per kilogram of the egg white; wherein the pool ofegg white has a volume of at least about 50 liters; mixing the acidicbuffer and the pool of egg white to form a mixed egg white having a pHfrom about 5 to about 6.5, wherein the mixed egg white comprises aprecipitant and a supernatant comprising the recombinant therapeuticprotein; and separating the precipitant from the supernatant comprisingthe recombinant protein.
 52. The method of claim 51, wherein the acidicagent is selected from the group consisting of acetic acid, hydrochloricacid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid,hydrobromic acid, perchloric acid, citric acid, boric acid, tartaricacid, lactic acid, formic acid, oxalic acid, uric acid, and barbituricacid.
 53. The method of claim 51, wherein the acidic buffer furthercomprises from about 5M to about 6M sodium acetate.
 54. The method ofclaim 51, wherein the acidic buffer is from about 0.5 wt % to about 2 wt% per kilogram of the pool of egg white.
 55. The method of claim 51,wherein the acidic buffer has a pH from about 4 to about 6.5.
 56. Themethod of claim 51, wherein the pH of the mixed egg white is from about5 to about 6.3.
 57. The method of claim 51, wherein the pool of eggwhite is mixed with the acidic buffer for at least about 1 hour.
 58. Themethod of claim 51, wherein the pool of egg white is mixed with theacidic buffer at a temperature from about 2° C. to about 25° C.
 59. Themethod of claim 51, wherein the pool of egg white has a volume of about100 liters to about 20,000 liters.
 60. The method of claim 51, whereinthe acidic buffer is added to the pool of egg white in a single bolusinjection.
 61. The method of claim 60, wherein the acidic buffer isadded at a rate of at least about 1 L/minute.
 62. The method of claim51, wherein the addition of the acidic buffer and the mixing of the poolof egg white are performed concurrently.
 63. The method of claim 51,wherein the separating step comprises filtering the mixed egg whitewithout allowing the mixed egg white to settle, wherein the filteringstep comprises filtering the mixed egg white through a filter having anaverage pore size ranging from about 0.1 μtm to about 100 μm.
 64. Themethod of claim 63, wherein the filtering step comprises one or moresubsequent filtering steps following an initial filtering of the mixedegg white, the one or more subsequent filtering steps using one or morefilters having an average pore size ranging from about 0.1 μm to about40 μm.
 65. The method of claim 51, further comprising the steps of:passing a pre-treatment buffer having a conductivity between about 8mS/cm and about 20 mS/cm through a filter; and passing the mixed eggwhite having a pH from about 5 to about 6.5 through the filter to obtaina filtered egg white.
 66. The method of claim 65, wherein thepre-treatment buffer has a pH from about 5.9 to about 6.1.
 67. Themethod of claim 65, wherein the pre-treatment buffer comprises sodiumphosphate and sodium chloride.
 68. A recombinant protein isolated fromegg white according to the method of claim
 51. 69. The method of claim51, wherein the conductivity of the mixed egg white is from about 8mS/cm to about 20 mS/cm.
 70. The method of claim 51, wherein therecombinant protein comprises a human lysosomal enzyme.